Molecular-based techniques involving the amplification of nucleic acids are increasingly being used in forensics, law enforcement, the military, human medicine, veterinary medicine, and research. In forensic, military and mass disaster situations, for example, DNA samples are now routinely taken from living persons thought to be relatives of unidentified victims of accident or foul play, to aid in identification of the dead. Military personnel or other individuals who expect to encounter hazardous situations where their lives may be at risk may wish to store DNA samples prior to exposing themselves to these hazards. In the law enforcement area, convicted felons in both Canada and the United States are now required to provide DNA samples. The use of DNA-based tests is expected to increase in medicine, for example, in testing for cystic fibrosis, cytochrome P450 isotypes, polymorphisms affecting susceptibility to infectious and autoimmune diseases, HLA typing, paternity issues, to name but a few. In clinical studies, an example would be to screen populations for colon cancer-predisposing genes or family members of a breast cancer victim for breast cancer predisposing genes. One technique for the amplification of DNA is the polymerase chain reaction (PCR).
PCR is a rapid, inexpensive and relatively simple means of amplifying copies of DNA molecules from a variety of source materials. However, a limitation of PCR is that DNA source materials typically contain a variety of inhibitors, such as pigments, proteins, saccharides and/or other impurities that interfere with the amplification reaction. For example, a variety of DNA polymerases, including Taq DNA polymerase (a typical thermostable DNA polymerase derived from Thermus aquaticus) are known to be inhibited by traces of body fluid-derived impurities in a PCR mixture. To overcome the problem of inhibitors within the DNA source material, there is typically a requirement for the purification of the DNA from the source material prior to amplification. However, purification procedures often involve multiple steps that can be time-consuming and expensive.
DNA can be extracted from nearly every type of cell in the human body and from a variety of cell-containing bodily fluids. The term “bodily fluid”, as used herein, can refers to a naturally occurring fluid from an animal, such as saliva, sputum, serum, plasma, blood, urine, mucus, gastric juices, pancreatic juices, semen, products of lactation or menstruation, tears, or lymph. A typical source of bodily fluid for extraction of DNA is white blood cells in venous blood. However, the use of blood as a source of DNA has many disadvantages. Collection of blood is not a trivial procedure. Taking of venous blood requires trained personnel. Furthermore, it is an invasive procedure, which frequently causes a degree of distress and pain to the donor. Precautions are needed to minimize exposure of blood collecting personnel to blood-borne pathogens. Once collected, the blood sample must be either frozen or quickly transported to a laboratory for extraction of DNA. A simpler procedure for obtaining blood is to collect a few drops after a finger prick and blotting it onto a piece of filter paper. Less training of personnel is required. Once dried, the DNA is quite stable. The amount of DNA recovered is small but sufficient for many forensic purposes. However, a finger prick is still an invasive procedure and haeme derived from haemoglobin in red blood cells can inhibit some types of DNA analysis.
Swabbing the inside of the cheek with a brush (a buccal swab) is another method of obtaining cells that contain DNA. This procedure is much less invasive than taking blood and permits collection by individuals with less training than is required in the collection of blood. Once collected, the time that useable DNA can be recovered is relatively short. Microbes in the mouth can degrade the DNA. However, this time can be extended by either drying the swab or wiping it onto filter paper and drying it.
Saliva is a fairly clear, colorless fluid secreted principally by the major salivary glands (parotid, submandibular, and sublingual). Its function is to lubricate and cleanse the oral cavity, as well as to initiate the process of digestion. The parotid gland primarily secretes serous (watery) saliva, while the other glands secrete a mixture of serous and mucinous (sticky) saliva. Components of saliva include mucins, and digestive enzymes.
Mucins are high molecular weight glycosylated proteins that form a major part of a protective biofilm on the surface of epithelial cells, where they can provide a barrier to particulate matter and bind microorganisms. These glycoproteins contribute greatly to the viscous nature of saliva.
It has long been known that cellular DNA is present in saliva and that this DNA is suitable for forensic purposes. Forensic use is typically limited to victim or suspect identification, using the small amounts of DNA from saliva that may recovered at a crime scene or from the back of a postage stamp. The notion that saliva may be a reliable source of genomic DNA and a rival to venous blood samples for this purpose has been investigated by van Schie, et al. (van Schie et al., (1997) J. Immunol. Methods. 208: 91-101). van Schie et al. used freshly collected or frozen saliva samples and purified the DNA by a fairly complex extraction procedure. Estimates of the quantity of DNA recovered were based upon light absorption at 260 nm, a procedure known to be an unreliable method since other common biological macromolecules, such as RNA, have essentially the same ultraviolet light absorption spectrum. Nevertheless, these authors showed that quality genomic DNA was indeed present by gel electrophoretic analysis and polymerase chain reaction analysis for certain allelic polymorphisms. Terasaki et al. (Terasaki et al. (1998) Hum Immunol. 59: 597-598) reported similar results about the suitability of saliva as a source of DNA for HLA typing by polymerase chain reaction analysis. Although the amount of DNA recovered was reported, the method used to measure DNA was not. These authors provided 3 examples where saliva dried on filter paper yielded DNA suitable for analysis.
There are significant advantages to providing a saliva sample rather than a blood sample as a source of DNA. Donors generally prefer donating saliva rather than blood because of the discomfort, pain, or apprehension associated with phlebotomy or pin-pricks. Saliva has a further advantage of not requiring specialized personnel thereby reducing cost where mass sample collection is being carried out. However, it will be clear to the skilled worker that while saliva is a preferred source of DNA, other bodily fluids, including blood, can be used.
More recently, it has been discovered that saliva can be used directly for real-time PCR without any DNA purification procedure. French et al. (French et al. (2002) Molecular And Cellular Probes. 16: 319-326) diluted fresh whole saliva 1:1 with water and used this mixture immediately, or following storage at 4° C. (2-3 days) or −20° C., for real-time PCR with a LightCycler instrument (Roche Diagnostics). PCR reaction volumes were typically 20 μl, containing 2 μl of saliva (diluted to 50% in water). The calculated concentration of DNA available for PCR was found to be between 0.1 ng/μl and 3.5 ng/μl, varying between samples obtained from different individuals and on different days. The authors commented that the amount of DNA available for amplification in crude saliva may not account for all of the DNA present in saliva samples, where a quantity of the total DNA may still reside within buccal cells or be too degraded to permit target amplification. A significant reduction in assay efficiency was not observed with saliva samples stored at 4° C. (2-3 days) or −20° C.
With the increasing use of DNA-based analysis in forensics, law enforcement, military, human medicine, veterinary medicine, and research, there is a need for compositions and methods that allow bodily fluids such as saliva to become a standard reliable source of DNA from an individual (to replace blood, the current standard). Desirably, it would be possible to use such compositions and methods for detecting DNA without requiring a separate step for extraction and purification of DNA from the saliva. Furthermore, it would be desirable to be able to store the bodily fluid at ambient temperature for several days. This would be especially advantageous when shipping of the saliva sample is required and/or a source of refrigeration is not available. In addition, it would be desirable if the concentration of genomic DNA in the saliva sample was high enough for both traditional PCR and real-time PCR without requiring additional steps. Traditional PCR usually requires DNA template in amounts >10 ng. According to the Roche Molecular Biochemicals PCR Application Manual (2nd edition, 1999, Roche Diagnostics), traditional PCR with low amounts of template (<10 ng human genomic DNA) requires special reaction modifications, such as changes in cycle number, redesign of primers, use of “Hot Start”, etc. In contrast, real-time PCR is much more sensitive than traditional PCR. For example, the LightCycler real-time PCR instrument (Roche Diagnostics) has 100% sensitivity for detecting 30 pg of control human genomic DNA (LightCycler Control Kit DNA manual, version 3, 2003).
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.